Fluorescent protein development 1) We are studying photoswitching behaviors of photoswitchable fluorescent proteins and their use in Frster Resonance Energy Transfer (FRET) experiments. These studies have led to the development of techniques and reagents to test new approaches to performing FRET measurements as well as developing new photoswitchable fluorescent proteins with the necessary characteristics to properly perform these experiments. 2) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques. As a consequence, we have commenced a project to survey numerous fluorescent proteins to better define their oligomerization characteristics. Our surveys of the literature have suggested these characteristics have not been rigorously determined using the proper ultracentrifugation analysis. Our hypothesis is that aberrant behavior observed when these proteins are tagged to some proteins of interest may be due to oligomerization. 3) We have an ongoing project to develop improved red fluorescent proteins. Current variants display low fluorescence, slow maturation, and/or oligomerization. Biochemical analyses of wild type proteins coupled with site-directed mutagenesis has led to our discoveries of mRuby variants with much decreased self-association, increased brightness, and faster maturation. 4) We have continued efforts to improve two-step fluorescent probes. The current standard, Padron, is one of the few available positive photoswitching probes and it lacks many of the characteristics of an optimal probe. Therefore, in addition to mutagenesis experiments to improve the folding and photoswitching behaviors of Padron, we are attempting to engineering positive photoswitching behaviors in other photoswitchable fluorescent proteins. Cell biology projects 1) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. Data have been collected on single expressed proteins using uninfected cells expressing PAFP tagged versions of the pUL37x1. Recent work has been performed using 2-color imaging to compare the localizations of multiple proteins of interest. 2) With Gaetan Herbomel, a post-doctoral fellow in the lab of Lawrence Tabak, we have undertaken a project to image the localization of Golgi apparatus enzymes using Stimulated Emission Depletion (STED) microscopy, Multifocal Structured Illumination Microscopy (MSIM), Photoactivated Localization Microscopy (PALM), and Stochastic Optical Reconstruction Microscopy (STORM). These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Specifically, we are interested in whether enzymes involved in early steps in sugar modifications of proteins are located in early compartments of the Golgi and vice versa. Instrumentation and imaging development 1) A total internal reflection fluorescence (TIRF) microscope system has been built by Yan Fu on an Olympus IX70 microscope obtained through property transfer. This instrument is been designed to provide a more homogeneous illumination pattern compared with traditional through objective TIRF by illuminating the sample from all angles possible for a through objective configuration. This is achieved by rapidly scanning the excitation beam in a circular pattern around the periphery of the objective rear aperture using galvanometers. By doing so, any diffraction patterns or other aberrations often found in TIRF imaging are averaged by imaging from various angles. We have developed a method to image at multiple positions with the TIRF excitation zone which effectively allows optical sections at 20-50 nm increment through the 200-300 nm illumination region.